Analysis of minute (sub-picomole) quantities of various organic molecules, as for example proteins, oligosaccharides, peptides, nucleotides, amino acids and DNA is of great value in many environmental, biotechnological, medical and pharmaceutical applications. In many cases, the available sample is very small, rendering analysis difficult and time consuming. Particularly this is the case where a small quantity of protein or peptide has been isolated and it is desired to identify the protein or peptide by determining the sequence of amino acids in the protein or peptide.
The basic chemistry of sequencing a peptide or a protein is known as Edman degradation chemistry, after P. Edman, Arch. Biochem. Biophys. 22, 475 (1979). In the Edman degradation reaction, the first step is a coupling step in which a peptide or protein (hereinafter described simply as a peptide) is first treated with a peptide degradation coupling agent such as phenylisothiocyanate (PITC), which couples to the peptide or protein to form a coupled peptide. The next step is a cleavage step in which the coupled peptide is treated with anhydrous acid, such as anhydrous trifluoroacetic acid (TFA), to cleave the coupled peptide to produce an amino acid residue, such as cyclic thiazolinone amino acid (ATZ), and leaving a truncated peptide, the peptide having had one amino acid residue cleaved from the peptide. In the next step, a conversion step, the amino acid residue is separated from the truncated peptide and treated with an aqueous solution or conversion agent, typically an aqueous acid such as aqueous TFA, to produce a converted amino acid residue, such as phenyl thiohydantoin (PTH) amino acid. The converted amino acid residue carries an amino acid that has been cleaved from the peptide. In the final step, identification, the cleaved amino acid is identified by some appropriate means.
An example of an apparatus for carrying out the Edman degradation reaction and sequencing a protein or peptide is described in R. Hewick et al, A Gas-Liquid-Solid Peptide and Protein Sequencer, The Journal of Biological Chemistry, Vol. 256, no. 15, Aug. 10, 1981, pp. 7990-7997. In the Hewick device as described in this paper, the sample peptide or protein is immobilized in a reaction chamber having a diameter of about 6 mm formed from a pair of facing conical cavities at the end of two facing glass rods. Capillaries, having diameter of about 0.5 mm, in the centers of the respective glass rods, supply reagent to and remove products from the reaction chamber. Coupling and cleavage are carried out in the reaction chamber and the derivatized amino acid residue is removed from the reaction chamber to a conversion flask, where the conversion step is carried out. The converted amino acid residue is then taken from the conversion flask, and the converted amino acid is identified by liquid chromatography. Further summary of the manner of operation of such an apparatus is described by M. W. Hunkapiller, in Protein/Peptide Sequence Analysis: Current Methodologies, A. S. Brown, ed., CRC Press Inc., Boca Raton La., 1988, at 87.
The entire degradation cycle of the Hewick apparatus requires in the order of 45 minutes, and has limited sensitivity. Further, the Hewick device requires relatively large quantities of reagent, which reduces its effectiveness for analyzing very small (femtomole, or 10.sup.-15 mole, or less) quantities of converted amino acid residue, in effect rendering it incapable of sequencing less than 1 picomole (10.sup.-12 mole) of peptide.
The inventors have proposed a novel reactor for reacting and subsequently analyzing sub-picomole quantities of a sample organic molecule. The reactor includes a continuous capillary connected between two valves that control fluid flow in the capillary. One part of the capillary forms a reaction chamber where the sample may be immobilized for subsequent reaction with reagents supplied through the valves. Another part of the capillary passes through or terminates in the detector portion of an analyzer such as an electrophoresis apparatus, liquid chromatographic apparatus or mass spectrometer.
The apparatus may form a peptide or protein sequencer for carrying out the Edman degradation reaction and analyzing the reaction product produced by the reaction. The protein or peptide sequencer includes a reaction chamber for carrying out coupling and cleavage on a peptide or protein to produce derivatized amino acid residue, a conversion chamber for carrying out conversion and producing a converted amino acid residue and means for identifying the converted amino acid residue. In one aspect of the invention, unlike in the Hewick device, the reaction chamber is contained within one arm of a capillary and the conversion chamber is located in another arm of the capillary. In a further aspect of the invention, an electrophoresis length of capillary is directly capillary coupled to the conversion chamber to allow electrophoresis separation of the converted amino acid residue as it leaves the conversion chamber. Identification of the converted amino acid residue takes place at one end of the electrophoresis length of the capillary.
In one aspect of a method according to the invention, the Edman degradation reaction is carried out in a capillary. Immobilization, cleavage and coupling of the peptide/protein takes place in a reaction chamber portion of the capillary to produce derivatized amino acid residue, and conversion takes place in a conversion chamber portion of the capillary to produce a converted amino acid residue. Electrophoresis separation of the converted amino acid residue then preferentially takes place in an electrophoretic length of the same capillary, including by applying an electric field across the conversion chamber, followed by identification of the converted amino acid residue.